This invention relates to a method for introducing an agent into the cytosol or plasma membrane of a cell using a lipid-coated tip of a pipette or rod or the like to contact, but not penetrate, the cell. In addition, the invention relates to a pipette or rod having a lipid-coated tip for introducing agents into the cytosol or plasma membrane of a cell and also to a method of coating a tip of a pipette or rod with the lipid.
The micro-injection of agents such as proteins and nucleic acid (mRNA and DNA) into the cytosol of living cells is a powerful technique which has led to many new discoveries. Furthermore, it underlies some new approaches to molecular and genetic engineering and is becoming useful in in vitro fertilisation. However, micro-injection by the insertion of a glass micro-pipette into the cell is a potentially damaging event and is limited, at present, to cells which are both large and robust. Micro-injection of small mammalian cells (spherical diameter =2-15 xcexcm) or very flat cells (1-2 xcexcm thickness) has always been difficult to achieve without damaging the cell and resulting in poor cell survival. The penetration of small cells, especially spherical cells, requires a rapid entry and exit xe2x80x9cstabxe2x80x9d, as slow withdrawal of the micropipette often results in breaking the cell. Thus xe2x80x9cstab injectionxe2x80x9d, where the micro-injector is within the cell for approximately 100 msec, high pressure (100-200 mbar) has to be used to introduce sufficient material into the cell during that time. This must be carefully controlled as insufficient pressure results in too little material being injected and excessive pressure causes cell damage or rupture. The cell must also be firmly attached to a substrate for the xe2x80x9cstabxe2x80x9dto effectively penetrate the cell membrane. Apart from these problems, the process is essentially blind and, because of the small size of the cell and the large volume of the cell cytoplasm occurred by organelles such as the nucleus, lysosomes and ER, insertion of the micro-injector at high speed is likely to cause intracellular damage. For example, the micro-pipette entering the cell with a velocity of approximately 700 xcexcm/sec is likely to displace, damage or enter the nucleus (which in small cells such as neutrophils and basophils accounts for up to 50% of the cell volume) rather than enter directly into the cytosol even if the plasma membrane survives.
Traditional pressure injection has been successfully used in a number of cell-types, including, hepatocytes (Cobbold) and Jurkat cells. However, the success rate is limited, with many cells failing to survive stab injection. There have been various attempts to minimise the problem of cell damage, including the use of pharmacological Ca2+ channel blockers. Other techniques have also been used for introducing agents, such as peptide inhibitors, antibodies etc into cells, by membrane permeabilisation using chemical or biological molecules, or techniques such as by electroporation. While this approach can be useful, cell damage is great, and mechanisms by which the cell repairs the damage are poorly understood, but which may involve Ca2+ and active signalling by the cell. Thus the recovered cells may not represent those in a state that was originally intended for study. Another method which has been used is internal perfusion. In this technique, the micro-pipette does not enter the cell, but membrane is sucked into the mouth of the micro-pipette causing a local rupture of the cell membrane at that point. This minimises damage to intracellular structures, but the process of xe2x80x9cbreaking intoxe2x80x9d the cell results in an irreversible seal and the cell must remain attached to the pipette for its survival. This precludes studies of cell shape change etc, and the negative pressure required to break-in also withdraws cytosol if not carefully controlled. In any case, during the period that the micro-pipette is attached diffusion of material both from the micro-pipette into the cell and from the cytosol into the micro-pipette occurs. Diffusion out of the cell of important small molecules such as GTP, ATP etc essentially limits the usefulness of this technique to those processes in which they are not involved or are regulated by the contents of the micro-pipette. All these techniques, while useful, thus have limitations.
Other approaches which do not involve glass micro-injection have also been used. One early approach was the use of lipid fusion, either by the use of liposomes or erythrocyte ghost function. While lipid fusion can be successful for introducing cDNA etc into cells as with lipofection, the amount of material injected is very small and perhaps only one copy of the cDNA is sufficient for the experiment. For example to inject a small cell (d=10 xcexcm) with 1% of its volume by fusion of liposomes (d=50 nm) requires thousands of fusion events. This number of fusion events is unlikely and the use of liposomes for cell physiology has not been widely successful. Fusion of larger vesicles such as red cell ghosts has also been used, where one fusion event can introduce a large amount of material. However, a large amount of foreign membrane is also introduced and the cells are essentially hybrids rather than original cells.
According to a first aspect of the present invention, there is provided a method for introducing an agent in to the cytosol and/or plasma membrane of a cell, comprising the steps of:
(a) coating at least a portion of a tip of a transferring apparatus with a lipid;
(b) bringing the lipid-coated tip of the transferring apparatus into contact with the cell; and
(c) transferring at least some of the contents of the transferring apparatus into the cytosol and/or plasma membrane of the cell without entering the cytoplasm.
The transferring apparatus is preferably a pipette or rod. The pipette is usually a micro-pipette (usually manufactured of glass) and the rod is usually a micro-rod. The micro-rod is a device (usually made of glass) which is solid and has no internal bore. A micro-pipette may be used for lipid-assisted micro-injection of substances into the cytosol or into the plasma membrane, whereas a micro-rod may be used to introduce substances exclusively into the plasma membrane. The micro-rod has the advantage that lipids and lipid-soluble molecules such as proteins can be transferred from the micro-rod to the cell and vice versa without aqueous transfer into the cytosol. In the case of a rod, the xe2x80x9ccontentsxe2x80x9d of the transferring apparatus may be present in the lipid on the tip.
It has been found that the technique provides fusion between the lipid at the tip and the cell membrane which results in a channel into the cell cytosol, without the possibility of intracellular organelle damage, and low pressure in the transferring apparatus ensures that the amount of material injected is controlled and does not unduly damage the cell. Furthermore, as only contact, rather than penetration, is required for agent introduction with this approach, the cell need not be firmly adherent. The applicants have carried out the use of simple lipid assisted micro-injection (xe2x80x9cSLAMxe2x80x9d) on human neutrophils, when loosely adherent as spherical individual cells or spread on glass coverslips with a cell thickness of just 1-3 xcexcm. These cells have been very difficult, if not impossible, to undergo successful micro-injection by conventional means.
Preferably, at least some of the contents of the transferring apparatus are transferred under pressure, which pressure is small enough to prevent damage to the cell contents. This pressure may typically be in the range of between 5 and 40 mbar.
The method is particularly useful when the cell is a living cell which remains living after transferring the contents of the transferring apparatus into the cytosol and/or plasma membrane of the cell. For example, the cell may be a small mammalian cell. Thus, the method of the invention has particular use in introducing agents in to a cell having a spherical diameter of between 2 and 15 xcexcm, or a cell of substantially flat form having a thickness of only 1 to 3 xcexcm. Thus, the cell may typically be a human neutrophil cell, having a thickness of 1 to 3 xcexcm.
Typically, transfer of the lipid and lipid-soluble molecules takes place between the transferring apparatus and the cytosol and/or plasma membrane of the cell when the contents of the transferring apparatus are transferred to the cell. The contents of the transferring apparatus are preferably in the form of an aqueous solution which may include a dye, for example lucifer yellow, which may aid in investigating transfer of the aqueous contents into the cell.
The method may further comprise the step of swelling the lipid to form a lipid-coating or bi-molecular layer (bilayer) prior to bringing the tip into contact with the cell.
In the present context, a lipid is any substance of a fat-like nature. Thus, the expression includes fatty acids or derivatives, which are soluble in organic solvents and insoluble in water, for example the simple fats and waxes and the phospholipids and cerebrosides. The expression also includes such compounds as sterols and squalene. As examples, both natural and synthetic lipids, and phospholipids, could be used in the present invention. The lipid preferably comprises phosphatidylcholine-oleyl-palmitoyl (PCOP). The PCOP may be dissolved in any suitable solvent, for example chloroform, before application of the lipid to the transferring apparatus, prior to drying of the lipid.
According to a second aspect of the present invention, there is provided a transferring apparatus for introducing an agent into the cytosol and/or plasma membrane of a cell, comprising a lipid-coated tip capable of transferring at least some of the contents of the transferring apparatus to the cell without entering the cytoplasm.
As discussed above, the transferring apparatus may be a pipette or a rod.
The tip may be disposable. Thus, there may be provided a lipid-coated tip for use in the transferring apparatus.
According to a further aspect of the present invention, there is provided a kit for introducing an agent into a cell, the kit comprising an agent and a transferring apparatus for introducing the agent into the cytosol and/or plasma membrane of a cell, wherein the apparatus comprises a lipid-coated tip capable of transferring at least some of the contents of the apparatus into the cytosol and/or plasma membrane of the cell without entering the cytoplasm.
According to a further aspect of the present invention, there is provided a method of transfecting a cell, which comprises introducing into the cell a transfection agent, the method comprising the steps of:
(a) coating at least a portion of a tip of a transferring apparatus with a lipid;
(b) bringing the lipid-coated tip of the transferring apparatus into contact with the cell; and
(c) transferring at least some of the contents of the transferring apparatus to the cell. There is also provided a cell or product of a cell treated in accordance with the methods of the invention.
According to a further aspect of the present invention, there is provided a method of coating a tip of a transferring apparatus with a lipid, comprising applying a lipid solution to the tip of the transferring apparatus and evaporating the solvent from the lipid solution.
The method is particularly suitable for the mass production of lipid coated pipettes or rods. The solvent is preferably volatile. The contact of the transferring apparatus with the lipid solution can be achieved in any suitable way, examples being immersion in solutions of the lipid or by passage of the transferring apparatus through an aerosol spray of the lipid solution.
The lipid solution may, in one embodiment, be provided in an air-tight and light-tight container.
The method of coating the tip may further comprise the step of loading the transferring apparatus with an aqueous solution prior to applying the lipid solution. The method may further comprise the step of, placing the transferring apparatus in an aqueous solution such that the dried lipid swells to form a lipid-coating or bi-molecular layer.
According to a further aspect of the present invention, there is provided a packaging for a transferring apparatus having a lipid coated tip, the packaging comprising an air-tight sheath member for receiving the transferring apparatus, wherein, when the packaging is in use, the sheath member surrounds at least the lipid-coated tip such that preservation of the lipid in its molecular form is maximised.
The packaging preferably further comprises an outer packaging member. The packaging may additionally further comprise a water-absorbing agent within the outer packaging member, for example silica gel. Preferably, the outer packaging member is flushed with an inert gas, for example nitrogen, before sealing thereof.
According to a further aspect of the present invention, there is provided a method of preparing a lipid-coated tip of a transferring apparatus, comprising:
(a) positioning a tip of a transferring apparatus in a sheath having an aperture therein;
(b) immersing the tip in a lipid in a solvent thereby allowing the solvent to pass through the aperture; and
(c) evaporating the solvent.
The aperture may be positioned in a base and side wall of the sheath, which is preferably removable.
According to a further aspect of the present invention, there is provided a method of preparing a transferring apparatus having a lipid-coated tip for use in introducing an agent in to the cytosol and/or plasma membrane of a cell, the method comprising the steps of:
a) placing the substance to be introduced in to the transferring apparatus;
b) immersing the tip of the transferring apparatus in an aqueous liquid surrounding the cell; and
c) applying a pressure to the contents of the transferring apparatus.
Preferably the substance to be introduced is placed in to the transferring apparatus from the rear (i.e. the non-lipid-coated end). A bi-molecular lipid layer may form on the transferring apparatus as it is immersed in the aqueous liquid. In step c), preferably a high pressure is applied, which may be between 1000 and 3000 millibar. The pressure is preferably applied transiently (eg. 0.1 to 2 secs). When the application of the pressure is stopped, the lipid at the tip reforms and the transferring apparatus is ready for use in lipid assisted micro-injection techniques as described.
Whilst the invention has been described above it extends to any inventive combination of the features set out above or in the following description.